Procaine salts of biosynthetic penicillins



Patented Oct. 31, 1950 g l 2,528,174

UNITED STATES PATENT OFFICE PROCAINE SALTS OF BIOSYNTHETIC PENICILLINS Harley W. Bhodehamel, Jr., Indianapolis, Ind., assignor to Eli Lilly and Company, Indianapolis, Ind., a corporation of Indiana No Drawing. Application July 1, 1948,

. Serial No. 36,455

4 Claims. (Cl. 260-302) 1 2 My invention relates to salts of biosynthetic are mixed together in amyl acetate to yield as a penicillins and more particularly to insoluble precipitate, the desired procaine salt of the biosalts of thenylpenicillins. synthetic penicillin. Alternatively, the sodium An object of my invention is to prepare insolsalt of the biosynthetic penicillin and procaine uble salts of thenyl penicillins. Another object '5 hydrochloride are reacted in aqueous solution to is to provide insoluble salts useful in the isolayield the insoluble procaine salt. The insoluble tion and purification of biosynthetic penicillins. salt which precipitates is isolated, as by filtration, Another object is to provide insoluble salts of and freed from traces of solvent in any suitable thenyl penicillins which have a prolonged theramanner, such as subjecting the salt to a vacuum. peutic effect. Other objects will be apparent from It is desirable to employ an amount of procaine the following disclosure of my invention, salt or procaine base in slight stoichiometric ex- I have discovered that thenyl penicillins form cess to assure the complete reaction of all of the crystalline salts with procaine and that such biosynthetic penicillin present. The procaine saltspossess properties whereby the above and salts of this invention being substantially inother objects are accomplished. soluble in water and the common organic sol- The novel salts of my invention may be repre- V vents, precipitate when prepared in one of these sented by the following formula solvents, leaving the excess procaine or soluble t RONHCHCH C(CHa)2 I-NCH.H2N o 0 o (0H1)2N(02H5)2 J) (30011 wherein R represents a member of the group consalt thereof in solution. It is believed that the sisting of the thenyl radical, lower alkyl-substi-' 5 procaine salt of the biosynthetic penicillin comtuted thenyl radicals, and halogen-substituted prises the combination of the acidic carboxyl thenyl radicals. group of the penicillin with the basic nitrogen Illustrative examples of compounds within the of the diethylaminoethyl group in the procaine scope of my invention are the procaine salts of molecule.

Z-thenyl penicillin, 4-chloro-3-thenyl penicillin, The biosynthetic penicillin may be recovered 4-ethyl-2-thenyl penicillin, 4-bromo-2-thenyl from its combination with procaine by dissolving penicillin and 4-propyl-3-thenyl penicillin. or suspending the procaine salt in aqueous solu- The thenyl penicillins used in producing the tion, acidifying the solution to about pH 2, and salts of my invention may be obtained by growextracting the biosynthetic penicillin with a ing a penicillin-producing mold in a culture penicillin "solvent such as amyl acetate, chloromedium containing a suitable precursor comform and the like. From the solution thus obpound which the mold may metabolize and incortained the biosynthetic penicillin may be recovporate in the biosynthetic penicillin. Procedures ered by any of the several suitable known for the preparation of the biosynthetic penicilprocesses. lins are described in detail in the publication by r 40 The following examples described specific Behrens et al., J. Biol. Chem., 175, 793 (1948). preparations of several salts of my invention.

The above-mentioned procaine salts of thenyl penicillins may be prepared by chemical reactions of addition or metathesis. The acidic form The procaine salt of 2-thenyl penicillin repreof the biosynthetic penicillin may be reacted with sented by the formula Example 1" procaine base, or a soluble metallic or.metalline is obtained as follows: v

(e. g. ammonia) salt of the biosynthetic penicillin Z-thenyl penicillin used in preparing the above may be reacted with a soluble acid addition salt salt may be produced by growing a penicillinof procaine. Thus, for example, the acidic form producing mold in a culture medium containing of the biosynthetic penicillin and procaine base about 0.02 percent of N-(Z-hydroxyethyD-Z- 3 4 thienylacetamide, and the biosynthetic penicillin containing about 2.5 g. of procaine base. The produced in the broth may be isolated as the procaine salt of Z-thienyl penicillin precipitates in sodium salt. crystalline form. The solution is cooled to about To about 5.2 parts of the sodium salt of the 2- C. for about 2 hours and is then filtered to isothenyl penicillin dissolved in about 30 parts of 5 late the precipitated procaine salt of Z-thienyl Water is added a solution of about 5 parts (20 penicillin. The isolated crystalline salt is washed percent excess) of procaine hydrochloride in 15 with a small amount of cold amyl acetate and parts of water. An immediate crystalline predried in vacuo to remove traces of moisture and cipitate of the procaine salt of 2-thenyl penicilresidual acetate. lin separates. The mixture is cooled to about The Z-thienyl penicillin is recovered from its 0 C. for about 2 hours and is then filtered to sepainsoluble salt as follows: 2 g. of the procaine salt rate the insoluble procaine salt of Z-thenyl peniare suspended in about 50 ml. of cold water and cillin. The separated salt is washed with a small sufiicient dilute sulfuric acid is added slowly to amount of cold water and is then dried in vacuo. acidity the solution to about pH 2. The acidified Example 2 mixture is extracted with about 50 ml. of amyl acetate whereupon the 2-thenyl penicillin dis- The procaine salt of 3-thenyl penicillin represolves in the amyl acetate, and the procaine in sented by the following formula the form of its sulfate salt dissolves in the aqueous may be prepared as follows: phase. The amyl acetate layer is separated from 3-thenyl penicillin is obtained by growing a the aqueous layer and the 2-thenyl penicillin is penicillin-Producing mold in a Cu u e medium recovered in purified form from the amyl acetate cont n n about p r of by extraction of the amyl acetate with cold dilute ethyl-3-thienylacetamide, and is isolated as its aqueous 11 1i 7 sodium salt. The procaine salt of B-thenyl peni- By proceeding through the insoluble procaine cillin is obtained from equal proportions of the penicillin salt, Substantial p rification f the sodium Salt of 3-l1hBny1 Penmimn and procame Z-thenyl penicillin is efiected, the degree of purihydroChlOTide by the Procedure described m fication amounting to 25 percent or more. If a Example 1. more greatly purified -2-thenyl penicillin is de- E fi ple 3 sired, the procaine salt of the penicillin is recrys- Instead of using a soluble salt of the penicillin tallized from alcohol or other suitable solvent and a procaine salt as described in Examples 1 prior to the recovery of the biosynthetic penicillin and 2, the reaction of the acid form of the biotherefrom. Recovery of the -2-thenyl penicillin synthetic penicillin and the procaine base may be from the purified procaine salt yields the biosynused. The biosynthetic penicilin in acid form is thetic penicillin in substantially pure form. obtained by extraction of the acidified broth.

Example 4 Thus, the procaine salt of Z-thienyl penicillin I v I I represented by the following formula The procaine Salt of 5-chlorocz-thenyl D 0 COOH may also be prepared as follows: Z-thienyl penicillin which may be represented by the following cillin is obtained by growing apenicilling-producformula OOH ing mold in a culture medium containing about is zbtailnethas gollgws: m I l 1 Y pe 101 inro ucing mo is grown in a cu plcent of N'mTmdmXyethyP ture medium containing about 0.02 percent of acetamlde and extractmg the cultme medium 9 the sodium salt of 5-chloro-2-thienylacetic acid, broth with amyl ac The amyl acetate 15 and the 5-chloro-2-thenyl penicillin produced in clarified with about 0.5 percent of decolorizing the broth is isolated as its potassium salt. carbon or fullers earth and is concentrated by T0 about 0119 p Of the Potassium Salt of the evaporation in vacuo. To about 4 liters of this m mT TY Y penicillin disiolved in about 6 parts of water is added a solution of about one t acetate m t about 3 of part of procaine hydrochloride in 3 parts of water. z'thlenyl 1381110111111 m acldlc form are added The crystalline precipitate of the procaine salt Slowly and with Stirring 10D Of a yl aceta e of 5-chloro-2-thenyl penicillin which separates is r 6 removed y filtration, l With a Small novel procaine salts of this invention should be amount of cold Water and drled 1I1 Vaclloused within a comparatively short time after their Example 5 preparation. For this reason it is often desirable to provide the procaine salts in oleaginous j procame salt of m 5 vehicles. A suitable oleaginous composition cillin represented by the following formula OOH may be prepared from the sodium salt of 4-methyl-3-thenyl penicillin and procaine hydrochloride which will retain therapeutic potency over a long according to the procedure described in EX- period of time is prepared by suspending in about ample 4. 5 ml. of sesame oil 2 g. of the procaine salt of 2- The 4-methyl-3-thenyl penicillin used in the thenyl penicillin, ground to a sufficiently small preparation can be obtained by growin a peniparticle size so that all of the material will pass cillin-producing mold in the presence of about rough a Z -me h n- In muscul r in- 0.02 percent of N-(Z-hydroxyethyl) -4-methyl-3- jection of 1 ml. ofthis suspension will maintain thienylacetamide, a therapeutically efiective blood concentration of For therapeutic purposes, the procaine salts y Penicillin for at least 4 hoursof the biosynthetic penicillins of this invention It 18 to i underst9qd that 9 above-meniloned preferably are administered intramuscularly as therapelltlc 00mD0S11'1'10I1S are glven y y Of finely divided suspensions of the salts in liquid ve- 111Strati9n Only n 1t W111 be pp t 3 6 hicles. Suitable vehicles for the provision of skilled i t art that numerous m0d1ficat10ns injectable suspensions include aqueous and olef Varlatlons 0f the above medlcmal 0011111081- aginous vehicles, for example, water, and vege- 1110115 y be madetable oils such as cottonseed oil, sesame oil, and I claim as my inventioni the like. As will be readily understood, the parl. The procaine salt of a thenyl penicillin repticle size of the salt in suspension should be suffiresented'by the formula ciently small to permit the particles to pass easily wherein R. represents a member of the group conthrough the lumen of a hypodermic needle withsisting of the thenyl radical, W y -S out packing within and blocking the needle. 11- stituted thenyl radicals, and halogen-substituted lustratively, a particle size which avoids blocking thenyl radicals. of a 20-gauge needle is that which will pass 2. The procaine salt of 2-thenyl penicillin repthrough a ZOO-mesh screen. 40 resented by the formula OOH By way of illustration, an aqueous medicinal 3. The procaine salt of 3-thenyl penicillin-repcomposition which upon intramuscular injection resented by the formula OOH is capable of maintaining a therapeutically effec- 4. The procaine salt of 5-chloro-2-thenyl penitive biosynthetic penicillin blood level for a period cillin represented by the formula OOH of about 24 hours comprises a suspension containing about 300 mg. of finely divided procaine HARLEY RHODEHAMEL salt of 2-thenyl penicillin per ml. of water. REFERENCES CITED Since, as is known, penicillins are relatively unstable in the presence of appreciable amounts The f01 1owmg references are of record m the of moisture, aqueous compositions containing the me of thls patent Drug and Cosmetic Industry, November 1947,

Page 595.

Certificate of Correction Patent No. 2,528,174 October 31, 1950 HARLEY W. RHODEHAMEL, JR.

It is hereby certified that error appears in the printed specification of the above numbered patent requiring correction as follows:

Column 1 line 52 for the word ammonia read ammonium' column 3 7 3 7 lines 44 54, and 74, and column 4, lines 2, 5, and 10, for 2-thienyl read -thenyl;

and that the said Letters Patent should be read as corrected above, so that the same may conform to the record of the case in the Patent Ofiice,

Signed and sealed this 26th day of December, A. D. 1950.

THOMAS F. MURPHY,

Assistant Uommz'aaz'oner of Patents. 

1. THE PROCAINE SALT OF A THENYL PENICILLIN REPRESENTED BY THE FORMULA 